Fluorescent-Based Quantitative Measurements of Signal Transduction in Single Cells

Abstract

Budding yeast (Saccharomyces cerevisiae) has been widely used as a model system to study fundamental biological processes. Genetic and biochemical approaches have allowed in the last decades to uncover the key components involved in many signaling pathways. Generally, most techniques measure the average behavior of a population of cells, and thus missed important cell-to-cell variations. With the recent progress with fluorescent proteins, new avenues have been opened to quantitatively study the dynamics of signaling in single living cells. In this chapter, we describe several techniques based on fluorescence measurements to quantify the activation of biological pathways. Flow cytometry allows for rapid quantification of the total fluorescence of a large number of single cells. In contrast, microscopy offers a lower throughput but allows to follow with a high temporal resolution the localization of proteins at sub-cellular resolution. Finally, advanced functional imaging techniques such as FRET and FCS offer the possibility to directly visualize the formation of protein complexes or to quantify the activity of proteins in vivo. Together these techniques present powerful new approaches to study cellular signaling and will greatly increase our understanding of the regulation of signaling networks in budding yeast and beyond. FLUORESCENT-BASED QUANTITATIVE MEASUREMENTS OF SIGNAL TRANSDUCTION IN SINGLE CELLS Serge Pelet and Matthias Peter Institute of Biochemistry, Department of Biology, ETH Zürich Abstract:Budding yeast (Saccharomyces cerevisiae) has been widely used as a model system to study fundamental biological processes. Genetic and biochemical approaches have allowed in the last decades to uncover the key components involved in many signaling pathways. Generally, most techniques measure the average behavior of a population of cells, and thus missed important cell-tocell variations. With the recent progress with fluorescent proteins, new avenues have been opened to quantitatively study the dynamics of signaling in single living cells. In this chapter, we describe several techniques based on fluorescence measurements to quantify the activation of biological pathways. Flow cytometry allows for rapid quantification of the total fluorescence of a large number of single cells. In contrast, microscopy offers a lower throughput but allows to follow with a high temporal resolution the localization of proteins at sub-cellular resolution. Finally, advanced functional imaging techniques such as FRET and FCS offer the possibility to directly visualize the formation of protein complexes or to quantify the activity of proteins in vivo.Together these techniques present powerful new approaches to study cellular signaling and will greatly increase our understanding of the regulation of signaling networks in budding yeast and beyond.

Effects of an indole derivative on cell proliferation, transfection, and alternative splicing in production of lentiviral vectors by transient co-transfection

“…The cells were incubated for 48 h and imaged using Cytation 1 Cell Imaging Multi-Mode Reader (BioTek CYT1FAV). Fluorescent microscopy is reported as a quantitative alternative method to flow cytometry [52,[76][77][78][79][80], provides comparable results [81], supports our economic, high-throughput, 96-well plate, adherent-culture workflow, and resulted in values within ranges reported for production of LV [82-84] (see Fig 2A).…”

Programmable Static Droplet Array for the Analysis of Cell–Cell Communication in a Confined Microenvironment

“…Haploid yeast of opposite mating type use reciprocal pheromones ( a - and α -factor) and receptors to signal cell cycle arrest, polarization, and eventually fuse to form a single diploid cell (Figure A) . To monitor α -factor-induced gene induction, we analyzed genetically engineered MAT a cells harboring a mating-specific reporter based on the FIG1 (factor induced gene 1) promoter driving the expression of the quadruple-Venus fluorescent protein . Cells were encapsulated in the SDA and exposed to synthetic pheromone in an array of 40 droplets (4 rows by 10 columns) by sequential droplet merging (Figure B).…”